DNA chip, PNA chip, and their preparation methods

ABSTRACT

A DNA chip (or PNA chip) composed of a solid carrier and plural DNA fragments (or PNA fragments) fixed onto the solid carrier at each one end, wherein a plurality of short chain spacer molecules having a hydrophilic moiety at each one end are fixed at each another end onto a surface of the solid carrier having no DNA fragments (or no PNA fragments) on its surface is effective for high sensitive quantitative analysis of a nucleic acid fragment complementary to the DNA fragment (or PNA fragment).

FIELD OF THE INVENTION

[0001] This invention relates to a DNA chip or a PNA chip favorablyemployable for detecting, with high sensitivity, a nucleic acid fragmentcomplementary to a DNA fragment or a PNA fragment of the DNA chip or thePNA chip, respectively The invention further relates to a method ofquantitative analysis of a nucleic acid fragment contained in a liquidsample in an extremely small amount using a DNA chip or a PNA chip whichhas a DNA fragment or a PNA fragment, respectively, on its solidcarrier.

BACKGROUND OF THE INVENTION

[0002] Detection of a nucleic acid fragment is generally made using aprobe DNA which is complementary to the nucleic acid fragment to bedetected, by way of hybridization. The probe DNA is generally fixed ontoa solid carrier (substrate) to give a DNA chip. In more detail, anucleic acid fragment in a sample liquid is once labelled with afluorescent label or a radioisotope label, and then the sample liquid isbrought into contact with the probe DNA of the DNA chip. If the labellednucleic acid fragment in the sample liquid is complementary to the probeDNA, the labelled nucleic acid fragment is combined with the probe DNAby hybridization. The labelled nucleic acid fragment fixed onto the DNAchip by hybridization with the probe DNA is then detected by anappropriate detection method such as fluorometry or autoradiography. TheDNA chip is widely employed in gene technology, for instance, fordetecting a complementary nucleic acid fragment or sequencing a nucleicacid.

[0003] The DNA chip is described, for instance, in Fodor S. P. A.,Science, 251, 767(1991) and Schena M., Science, 270, 467(1995). The DNAchip is understood to efficiently detect a small amount of acomplementary nucleic acid fragment in a small amount of a sampleliquid. However, the heretofore known DNA chip has a limitation on thedetection level of nucleic acid fragment such as a level of not lessthan 10⁻¹⁹ mol./dot. In certain cases, an improved high sensitivedetection is required. For instance, a procedure for monitoring of geneexpression, particularly gene expression of a low level, a procedure foranalyzing a gene variation, and a procedure for analyzing genepolymorphisrn, require higher sensitivity in the detection ofcomplementary nucleic acid fragments

[0004] Detection of nucleic acid fragment using an electrochemical labelis also known (Japanese Patent Provisional Publication No. 9-288080, anda preprint of the 57th Analytical Chemistry Conference pp. 137-138(1996)). The electrochemical label such as N-hydroxysuccinimide ester offerrocenecarboxylic acid is attached to a probe DEN. The probe DNA isfixed onto an electroconductive substrate having an output terminal. Inthe detection procedure, a sample liquid containing the target nucleicacid fragment is brought into contact with the probe DNA having theferrocene derivative label in the presence of an electrochemicallyactive thread intercalator. The target nucleic acid fragment, if it iscomplementary to the probe DNA, is hybridized with the probe DNA. Intothe formed hybrid structure, the electrochemically active threadintercalator is intercalated Thereafter, a potential is applied to theelectroconductive substrate to measure an electric current flowingthrough the ferrocene derivative label and the thread intercalator.

[0005] On the above-mentioned electroconductive substrate are fixed theferrocene derivative-modified DNA fragments of approximately 10⁻¹¹ mol.,per 1 mm² of surface area of the substrate). It is described thatcomplementary nucleic acid molecules in the range of 10⁻¹⁵ to 10⁻¹¹ mm²are detected, when a sample liquid containing target nucleic acidmolecules is brought into contact with the probe DNA.

[0006] Preprint of the 47th Polymer Society Conference, pp. 3155-3156(1998) describes an electrochemical detection method in which a sampleliquid containing a target nucleic acid fragment is brought into contacta DNA probe fixed onto an electroconductive substrate in the presence ofan electrochemically active thread intercalator On the electroconductivesubstrate are fixed the DNA probes in an amount of approximately 10⁻¹¹mol./2 mm² (surface area of substrate). If a sample liquid containingtarget nucleic acid fragments in an excessive amount (such asapproximately ten times or more) is brought into contact with the probeDNA, complementary nucleic acid fragments in an amount of approximately10⁻¹¹ mol. are detected.

[0007] P. E. Nielsen et al., Science, 254, 1497-1500(1991) and P. E.Nielsen et al., Biochemistry, 36, pp.5072-5077 (1997) describe PNA(Peptide Nucleic Acid or Polyamide Nucleic Acid) which has no negativecharge and functions in the same manner as DNA does. PNA has apolyarnide skeleton of N-(2-aminoethyl)glycine units and has neitherglucose units nor phosphate groups. A representative PNA as well as arepresentative DNA are illustrated below:

[0008] Since PNA is electrically neutral and is not charged in theabsence of an electrolytic salt, PNA is able to hybridize with acomplementary nucleic acid fragment to form a hybrid which is morestable than hybrid given by a DNA prove and its complementary nucleicacid fragment (Preprint of the 74th Spring Conference of Japan ChemicalSociety, pp. 1287, reported by Naomi Sugimoto).

[0009] Japanese Patent Provisional Publication No.11-332595 describes aPNA probe fixed on a solid carrier at its one end and a detection methodutilizing the PNA probe The PNA probe is fixed onto the solid carrier bythe avidinbiotin method.

[0010] The aforementioned P. E. Nielsen et al., Science, 254,1497-1500(1991) also describes a PNA probe labelled with isotope elementand a detection method of a complementary nucleic acid fragment.

[0011] Since the PNA probe shows no electric repulsion to a targetnucleic acid fragment in a sample liquid, an improved high detectionsensitivity is expected.

SUMMARY OF THE INVENTION

[0012] It is an object of the present invention to provide a nucleicacid fragment-detecting means showing an improved high sensitivity for anucleic acid fragment contained in a sample liquid.

[0013] Specifically, it is an object of the invention to provide a DNAchip and a PNA chip which are employable for quantitatively detecting anucleic acid fragment contained in a sample liquid in an extremely smallamount.

[0014] It is another object of the invention to provide a method forquantitatively detecting, with a high sensitivity and goodreproducibility, a nucleic acid fragment contained in a sample liquid inan extremely small amount.

[0015] The present invention resides in a DNA chip comprising a solidcarrier and a plurality of DNA fragments fixed onto the solid carrier ateach one end, wherein a plurality of short chain spacer molecules havinga hydrophilic moiety at each one end are fixed at each another end ontoa surface area of the solid carrier having no DNA fragments thereon.

[0016] The invention further resides in a PNA chip comprising a solidcarrier and a plurality of PNA fragments fixed onto the solid carrier ateach one end, wherein a plurality of short chain spacer molecules havinga hydrophilic moiety at each one end are fixed at each another end ontoa surface area of the solid carrier having no PNA fragments thereon.

[0017] The DNA chip (or PNA chip) of the invention is favorably preparedby a process comprising the steps of:

[0018] applying onto a solid carrier an aqueous solution of a pluralityof DNA fragments (or PNA fragments) dissolved or dispersed in an aqueousmedium to fix the DNA fragments (or PNA fragments) onto the solidcarrier; and

[0019] applying onto the solid carrier having thereon the fixed DNAfragments (or PNA fragments) an aqueous solution of short chain spacermolecules having at each one end a hydrophilic moiety and at eachanother end a moiety reactive to fix to the solid carrier

[0020] In the DNA chip and PNA chip of the invention, the solid carrierpreferably is an electro-conductive substrate.

[0021] The DNA chip (or PNA chip) of the invention is favorably employedin a method of quantitative analysis of a nucleic acid fragmentcontained in a sample liquid which is complementary to the DNA fragments(or PNA fragments) of the DNA chip (or PNA chip), which comprises thesteps of:

[0022] adjusting the concentration of the nucleic acid fragment in thesample liquid so that a droplet of the sample liquid applied to the DNAchip (or PNA chip) should contain 10⁻²⁰ to 10⁻¹⁶ mol. of the nucleicacid fragment per 1 mm² of the surface of the electro-conductivesubstrate of the DNA chip (or PNA chip);

[0023] bringing the nucleic acid concentration-adjusted sample liquidinto contact with the DNA chip (or PNA chip) having an electroconductivesubstrate, whereby hybridizing the nucleic acid with the DNA fragment(or PNA fragment) on the DNA chip (or PNA chip);

[0024] bringing an electrochemically active molecule in contact with thehybridized nucleic acid and DNA fragment (or PAt fragment), wherebyattaching the electrochemically active molecule to the hybridizednucleic acid and DNA fragment (or PNA fragment);

[0025] applying a potential to the DNA chip (or PNA chip); and

[0026] measuring an electric current flowing from or to theelectro-conductive substrate through the attached electrochemicallyactive molecule.

[0027] Accordingly, the DNA chip (or PNA chip) of the invention ispreferably supplied in the form of a kit for conducting quantitativeanalysis of a nucleic acid fragment contained in a sample liquid whichis complementary to the DNA fragments (or PNA fragments) of the DNA chip(or PNA fragment), which comprises the DNA chip (or PNA chip) having anelectroconductive substrate and an electrochemically active moleculewhich is attachable to a hybridized nucleic acid fragment and DNAfragment.

[0028] In the DNA chip (or PNA chip) of the invention, the DNA fragments(or PNA fragments) are preferably fixed on the solid carrier in anamount of 10⁻²⁰ to 10⁻¹² mol. /mm².

[0029] The hydrophilic moiety of the spacer molecule preferably is ahydroxyl group, and the spacer molecule is preferably fixed on the solidcarrier through a mercapto moiety attached to the end of the spacermolecule. The spacer molecule is preferably derived from a compoundselected from the group consisting of 2-mercaptoethanol,3-mercaptoethanol, 6-mercaptoetol, andN,N′-di(3-hydroxy-n-propyl)imidazole-2-thione. The spacer molecule maycontain a cyclic group in the molecular structure.

BRIEF DESCRIPTION OF DRAWINGS

[0030]FIG. 1 schematically shows a DNA chip (or a PNA chip) of theinvention and a mechanism of the detection of complementary nucleic acidfragments according to the invention.

[0031]FIG. 2 is a graph indicating an electric current observed in thedetection method of the invention as well as an electric currentobserved in the case that no hybridization occurs between the prove DNAand a target nucleic acid fragment.

DETAILED DESCRIPTION OF THE INVENTION

[0032] The DNA chip and PNA chip according to the invention aredescribed, in more detail, by referring to FIG. 1. Since the DNA chipand PNA chip have essentially the same structure, the followingdescription is mainly addressed to the structure and preparation of theDNA chip.

[0033]FIG. 1-(1) schematically illustrates a conventionally employed DNAchip 31 which comprises a solid carrier (e.g., electroconductivesubstrate) 11 and a plurality of DNA fragments 21 fixed onto the solidcarrier at each one end.

[0034]FIG. 1-(2) schematically illustrates a DNA chip 51 of theinvention which is prepared by bringing an aqueous solution containing aplurality of spacer compounds (or mask compounds, which has a shortchain structure, as compared with the chain structure of the previouslyfixed DNA fragment) 41 a into contact with the DNA chip 31 of FIG. 1-(1)so that the plural spacer compounds 41 a are attached to the solidcarrier 11 in the areas where the DNA fragments are not fixed. The fixedspacer compounds (namely, spacer molecules) 41 b have a hydrophilicgroup 42 on each of their free terminal ends.

[0035] [Solid Carrier]

[0036] The solid carrier of the DNA chip of the invention can be any ofknown solid carriers or their equivalent materials, for instance, anelectroconductive substrate (e.g., electrode), a plastic sheet, and aglass sheet. The electroconductive substrate is generally employed forconducting the electrochemical analysis.

[0037] The electroconductive substrate may be provided on anelectro-insulative support material. The electroconductive substrate andthe support material preferably have a less hydrophilic surface or ahydrophobic surface. The electroconductive substrate may have a plainsurface or a surface having many fine concaves and convexes.

[0038] The electro-insulative support material can be prepared fromglass, ceramics, polymer materials (e.g., polyethylene terephthalate,cellulose acetate, polycarbonate of Bisphenol A, polystyrene,poly(methyl methacrylate), silicon, active carbon, and porous materials(e.g., porous glass, porous ceramics, porous silicon, porous activecarbon, cloth, knitted cloth, non-woven cloth, filter paper, andmembrane filter). Polymer materials, glass, and silicon are preferablyemployed.

[0039] Generally, the electro-insulative support material is employed inthe form of a sheet (or a film) The sheet of the support materialpreferably has a thickness in the range of 100 to 1,000 μm.

[0040] The electroconductive substrate can be made of electrodematerial, optical fiber, photodiode, thermistor, piezo electricalelement, or surface elasticity element. The electrode material isgenerally employed. The electrode can be carbon electrode of graphite orglassy carbon, noble metal electrode of platinum, gold, palladium, orrhodium, metal oxide electrode of titanium dioxide, tin oxide, manganeseoxide, or lead oxide, semiconductor electrode of Si, Ge, ZnO, or Cds, orelectron conductor of titanium. Preferred are glassy carbon electrodeand gold electrode. The electrode may be covered with electroconductivepolymer film or monomolecular film

[0041] The DNA chip of the invention is preferably composed of ahydrophobic, electro-insulative support material, a plurality ofhydrophobic electroconductive substrates placed on the support material,a plurality of DNA fragments fixed on each of the electroconductivesubstrates, and a plurality of spacer molecules fixed on the substratesat free areas (i.e., areas where no DNA fragments are present). Each ofthe electroconductive substrates is preferably arranged apart from theadjoining electroconductive substrates so that each substrate isinsulated from the adjoining substrates. The electroconductive substratemay be placed on the support material via an intermediate layer such asa hydrophilic intermediate layer which may have electron charges.

[0042] An example of the structure composed of an electroinsulativesupport material and a plurality of electrodes arranged on the supportmaterial is a silicon chip described in Sosnowski, RG., et al, Proc.Natl. Acad. USA, 94, 1119-1123(1997). The electrode may be produced on apolymer film using a composite sheet of a polymer film and a metal film.

[0043] [DNA Fragment]

[0044] DNA fragment to be fixed onto the solid carrier may bepolynucleotide such as cDNA, a portion of cDNA, or EST. Thepolynucleotide is favorably employed for studying gene expression.Otherwise, DNA fragment to be fixed onto the solid carrier may beoligonucleotide, which is favorably employed for studying variations andpolymorphism of gene. The DNA fragment to be fixed onto the solidcarrier preferably is one of 3 to 50 mers, more preferably 10 to 25mers.

[0045] If the DNA chip of the invention comprises plural DNA chip unitseach of which has an electroconductive substrate (e.g., electrode) andDNA fragments fixed onto the substrate, the plural DNA chip units canhave the same DNA fragments or different DNA fragments.

[0046] Fixation of DNA fragments onto the substrate can be done by anyof known methods. For instance, DNA fragments having a reactive group onone end can be fixed onto the substrate through covalent bond by thereaction of the reactive group and the functional group of the surfaceof the substrate. For instance, a mercapto group is attached to DNAfragment at its 5′- or 3′-terminal, and the mercapto group is thencaused to react with a gold electrode, so that an electroconductivesubstrate having DNA fragments fixed thereon is produced. The procedurefor attaching a mercapto group to DNA fragments is described in M Maeda,et al., Chem. Lett., 1805-1808(1994) and B. A. Connolly, Nucleic AcidsRes., 13, 4484(1985).

[0047] If the electroconductive substrate is made of glassy carbonelectrode, the glassy carbon electrode is oxidized by potassiumpermanganate to produce a carboxylic acid group on the electrode. Thecarboxylic acid group on the electrode forms an amide bonding with DNAfragment so that the DNA fragment is fixed onto the substrate. See K. M.Millan, et al., Analytical Chemistry, 65, 2317-2323(1993).

[0048] DNA fragments can be fixed to the substrate, initially, in theform of hybrid DNA fragments. For instance, the hybrid DNA fragments arecombined with a mercapto group at their 5′- or 3′-terminals (preferably5′-terminals) of their single fragment, and are brought into contactwith a gold electrode, so that the hybrid DNA fragments are fixed on theelectrode. The hybrid DNA fragment fixed on the electrode is thenprocessed to dissociate a single fragment having no mercapto group, sothat the desired DNA chip is produced.

[0049] The covalent bond between the DNA fragment and the substrate canbe formed using an amino group, an aldehyde group, a mercapto group, ora biotin molecule which is attached to the DNA fragment. If thesubstrate is made of glass sheet or silicon sheet, its surface ispreferably treated with a silane coupling agent, prior to the formationof covalent bond.

[0050] Otherwise, DNA fragments can be synthesized on the substrate by aknown method.

[0051] The DNA fragment having a reactive group on one end can be fixedonto a solid carrier such as an electroconductive substrate by spottingonto the carrier an aqueous solution containing the DNA fragment Theaqueous solution preferably contains the DNA fragment in a concentrationof several pm to several mM The volume for the spotting generally is inthe range of 1 to 100 nL, preferably 1 to 10 nL. The aqueous solutionmay contain a viscosity increasing additive such as sucrose,polyethylene glycol, or glycerol The spotting can be made manually orutilizing a commercially available spotter. The spotted solution is thenkept on the solid carrier at a predetermined temperature for severalhours (namely, incubation), whereby the DNA fragment is fixed onto thecarrier by covalent bonding. After the incubation is complete, free DNAfragment (which is not fixed onto the carrier) is preferably washed out.

[0052] The DNA fragment is preferably fixed onto the solid carrier in anamount of 10⁻²⁰ to 10⁻¹² mol./mm² of the surface area of the carrier.The amount of the fixed DNA fragment can be determined by means of HPLC(high performance liquid chromatography) or other analyticalapparatuses.

[0053] [P Fragment]

[0054] The PNA fragment preferably employable in the invention has thefollowing formula (I):

[0055] In the formula, the symbols of B¹¹, R¹¹, L¹¹, a, b, c, and d havethe meanings described below.

[0056] B¹¹ is a ligand and represents one of bases of natural nucleicacids (i.e., A, T, C, G, I, or U) or its analogue. B¹¹ is bonded throughthe 9th position in the case that the base is a purine base such asadenine, guanine or inosine, and through the 1st position in the casethat the base is a pyrimidine base such as thymine, uracil or cytosine.The base analogue is an organic base which is similar to the base ofnatural origin in its chemical structure, for instance, a base groupwhich is prepared by replacing the carbon or nitrogen atom of the purineor pyrimidine ring with a nitrogen or carbon atom, respectively, or abase group modifying the purine or pyrimidine ring with a substituentsuch as a sulfhydryl group or a halogen atom. Otherwise, B¹¹ can be anaromatic moiety containing no nucleic acid base, an alkanoyl grouphaving 1 to 4 carbon atoms, a hydroxyl group, or a hydrogen atom.Examples of the base analogues include 7-deazaadenine, 6-azauracil, and5-azacytosine. B¹¹ also can be a DNA intercalator, a reporter ligand, aprotein label such as hapten or biotin, a spin label, or a radioactivelabel. Particularly preferred are nucleic acid bases (i.e., A, T, C, G,and U).

[0057] R¹¹ is a hydrogen atom or a group derived from a side-chain of anα-amino acid of natural origin. Examples of such groups include an alkylgroup having 1 to 6 carbon atoms, an aryl group having 6 to 20 carbonatoms, an aralkyl group having an alkyl group of 1 to 6 carbon atoms, aheteroaryl group having 6 to 20 carbon atoms, a hydroxyl group, analkoxy group having 1 to 6 carbon atoms, an alkylthio group having 1 to6 carbon atoms, a group of -NR¹³ R¹⁴ [each of R¹³ and R¹⁴ independentlyis a hydrogen atom, an alkyl group having 1 to 3 carbon atoms, an alkoxygroup having 1 to 3 carbon atoms, an alkylthio group having 1 to 3carbon atoms, or a hydroxyl group], and a mercapto group. R¹¹ may forman alicyclic ring or a heterocyclic ring in combination with the carbonatom to which R¹¹ is attached.

[0058] L¹¹ is a linking group such as a divalent group represented bythe group of —Co— or —CoNR¹²—[R¹² is a hydrogen atom, an alkylene grouphaving 1 to 4 carbon atoms, a hydroxyl group, an alkoxy group having 1to 4 carbon atoms, or an amino group], or an alkylene group having 1 to4 carbon atoms. The alkoxy group and amino group may have one or moresubstituents such as alkyl of 1-4 carbon atoms, alkoxy of 1-4 carbonatoms, and hydroxyl.

[0059] Each of a, b and c independently is an integer of 0 to 5,preferably 1, and d is an integer of 1 to 60, preferably an integer of 1to 40.

[0060] A particularly preferred PNA fragment has the following formula(II), in which each of B¹¹ and d has the same meaning as described abovefor the formula (I):

[0061] The PNA fragment can be fixed onto a solid carrier in a knownmanner (see Protein, Nucleic Acid, Enzyme, Vol.43, No.13,2004-2011(1988) and Japanese Patent Provisional Publication 9-288080.Also employable are procedures similar to the aforementioned DNAfragment-fixing procedures.

[0062] [Short Chain Spacer Molecules]

[0063] The DNA chip of the invention has a plurality of short chainspacer molecules having a hydrophilic moiety at each one end which arefixed at each another end onto a surface area of the solid carrierhaving no DNA fragments thereon, as schematically illustrated in theattached FIG. 1-(2). The coverage of the solid carrier using the spacermolecules can be expressed in terms of “masking treatment”.

[0064] The short chain of the spacer molecule means that the molecularlength of the spacer molecule is sufficiently short, as compared withthe length of the DNA fragment fixed onto the carrier in their vicinity.

[0065] The spacer molecule preferably has a main skeleton composed of analkylene group having 1 to 6 carbon atoms.

[0066] The spacer molecule may have a cyclic group in the molecularchain or as a substituent. Examples of the cyclic groups include an arylgroup having 6 to 12 carbon atoms, a cycloalkyl group having 3 to 6carbon atoms, and a heterocyclic group containing 1 to 4 hetero atoms(e.g., N, S, O, or P) and 2 to 20 carbon atoms. The cyclic group cancontain the reactive group which can be reacted with the carrier surfaceto fix the spacer atoms to the carrier, One example is aimidazole-2-thione group.

[0067] The hydrophilic moiety can be attached to the spacer molecule inthe terminal position or in the vicinity of the terminal position. Oneor more hydrophilic moieties may be attached to the spacer molecule.Examples of the hydrophilic moieties include hydroxyl, carboxyl, amido,phosphoryl, and sulfonyl Preferred is hydroxyl.

[0068] The other end of the spacer molecule is fixed onto a surface areaof the carrier where the DNA fragment is not attached. The fixation ofthe spacer molecule is preferably performed utilizing a reactive groupwhich is attached to the spacer molecule at the other end. Examples ofthe reactive groups include mercapto, sulfide, disulfide, thiocarbonyl,and thiocarboxyl Preferred are mercapto, thiocarbonyl, and sulfide Mostpreferred is mercapto. It is also preferred that the solid carrier has areactive group, on its surface, such as amino, imino, hydrazino,hydrazide, amide, carboxyl, aldehyde, epoxy, or peroxy.

[0069] Examples of the reactive compounds serving as the spacermolecules include mercaptomethanol, 2-mercapto-ethanol,3-mercaptopropanol, 4-mercaptobutanol, 5-mercaptopentanol,6-mercaptohexaol, N,N′-di(3-hydroxy-n-propyl) imidazole-2-thione, andvarious imidazole-2-thione derivatives described in A. J. Arduengo, etal., J. Am. Chem. Soc., 1990, 112, 6153-6154. Preferred are2-mercaptoethanol, 3-mercaptopropanol, 4-mercaptobutanol,5-mercaptopentanol, and 6-mercaptohexanol. Most preferred is2-mercaptoethanol. These active compounds may be in the form of theirsalts with sodium or potassium. The alkylene chain of the activecompound can be substituted with one or more substituents such as ahydrocarbyl group having 1 to 6 carbon atoms (e.g., methyl, ethyl, orphenyl).

[0070] Onto the solid carrier, two or more different spacer moleculescan be provided.

[0071] [Hybridization]

[0072] The hybridization can be performed essentially in the same manneras that employed in various assay procedures utilizing the conventionalDNA chip.

[0073] When the electrochemical analysis is performed, anelectrochemically active molecule, specifically, an electrochemicallyactive thread intercalator, is preferably employed for insertion into ahybrid formed by the DNA fragment and a sample nucleic acid fragment onthe electroconductive substrate. The thread intercalator assists easyflowing-of electric current from or to the substrate along the formedhybrid structure. The electrochemical thread intercalator can be presentwhen hybridization takes place. Otherwise, the thread intercalator canbe brought into contact with a previously formed hybrid structure. Inthe latter case, a free nucleic acid fragment which is not hybridizedwith the fixed DNA fragment is preferably removed from the substrate bywashing with-a mixture of a surfactant (preferably sodiumdodecylsulfate) and a buffer (preferably a citrate buffer) in advance ofthe contact with the intercalator. The intercalator is preferablybrought into contact with the hybrid in an aqueous solution at aconcentration of 10 nM to 10 mM.

[0074] The hybridization is preferably performed at a temperaturebetween room temperature and approximately 70° C., for 0.5 to 20 hours.

[0075] [sample Nucleic Acid Fragment]

[0076] The sample nucleic acid fragment can be a DNA fragment obtainedfrom a living sample, if necessary, after performing an appropriateprocedure, a DNA fragment obtained by cleaving a DNA fragment producedby gene technology and isolating the cleaved product by electrophoresis,a single stranded-DNA fragment which is synthesized by a chemicalprocess A double stranded DNA fragment is preferably treated with heator an alkaline solution to give a single stranded fragment. Plural kindsof nucleic acid fragments can be analyzed on one DNA chip

[0077] The sample nucleic acid fragment is brought into contact with DNAfragment fixed on the solid carrier in the form of its aqueous solution.The aqueous solution preferably contains the sample nucleic acidfragment at a concentration of several pM to several mM, preferably at aconcentration of several pM to several nM.

[0078] [Electrochemically Active Thread Intercalator]

[0079] The electrochemically active thread intercalators favorablyemployed for the electrochemical analysis of nucleic acid fragments arealready known. A representative example of the intercalator is a threadintercalator hating an electroconductive group at one end or both ends.The thread intercalator having the electroconductive group preferablyhas an oxidative-reductive activity. The oxidative-reductive activitycan be imparted to the thread intercalator by incorporating into theintercalator a ferrocene group, a catechol amine group, a metalbipyridine complex group, a metal phenathroline complex group, or aviologen group. The intercalator moiety preferably comprises anaphthaleneimide moiety, an antbracene moiety, or an anthraquinonemoiety Preferred electrochemically active thread intercalator is aferrocene-containing naphthalene diimide compound [NDIFc₂-1, which isprepared from carboxylic acid ester of N-hydroxysuccinimide and acorresponding amine compound, see S. Takenaka et al., J. Chem. Soc.Commun., 1111 (1998)]:

[0080] A ferrocene-containing naphthalene diimide derivative having thefollowing formula is also preferably employed:

[0081] In the above-illustrated formula: X is one of the followingferrocene derivative groups:

[0082] The thread intercalator having an electroconductive groupcomprises not only the oxidative-reductive active moiety and theintercalator moiety but also a linker moiety placed between thesemoieties. The 1,4-dipropyl-piperazinyl group of the formula is anexample of the linker moiety. The piperazinyl group can be replaced withan quaternary imino group. An intercalator of the below-illustratedformula which has a quaternary imino group always is cationic regardlessof pH condition. This means that the intercalator is firmly fixed to theDNA hybrid and PNA hybrid. Accordingly, it is favorably employed in theinvention. Particularly, the intercalator having a quaternary iminogroup is preferred in the use in combination with the PNA chip Thelinker can be an N-alkyl group having 1 to 6 carbon atoms (e.g, methyl,ethyl, or n-propyl). The oxidative-reductive potential of the ferrocenemoiety of the intercalator varies depending upon the nature of thelinker moiety.

[0083] [High Sensitive Quantitative Analysis of Sample Nucleic AcidFragment]

[0084]FIG. 1-(1), -(2), -(3), and -(4) schematically illustrate themechanism of the high sensitive quantitative analysis of sample nucleicacid fragment according to the invention. As described hereinbefore,FIG. 1-(2) illustrates a DNA chip (or PNA chip) 51 of the inventionhaving the spacer molecules 41 b on an electroconductive substrate 11between the fragments 21 The spacer group 41 b has a hydrophilic group(e.g., hydroxyl group) 42 on one end which is opposite to the other endat which the spacer is fixed to the substrate.

[0085] When an aqueous solution containing a sample nucleic acidfragment 61 which is complementary to the DNA fragment 21 of the DNAchip 51 is spotted onto the DNA chip, the sample nucleic acid fragment61 hybridizes with the DNA fragment 21 to give a hybrid double strandedstructure 71, as illustrated in FIG 1-(3)-If the hybridization isperformed in the presence of an electrochemically active threadintercalator 81 a, it enters within the hybrid structure 71 to give anelectroconductive portion 81 b, as illustrated in FIG. 1-(4). Theprocedures are generally performed in an aqueous medium. Under thiscondition, an electric potential is applied to the electroconductivesubstrate 11, whereby an electric current flows from or to theelectroconductive substrate 11 along the hybrid structure 71 having theelectroconductive portions 81 b. The electric current is then measured.The quantitative analysis of the invention is further described below inmore detail.

[0086] In the first step, an aqueous solution of a sample nucleic acidfragment is prepared. The sample nucleic acid fragment is dissolved ordispersed in an aqueous medium. The aqueous solution of sample nucleicacid fragment is then brought into contact with the DNA chip. Thecontact can be performed by spotting the aqueous solution on the DNAchip or by immersing the DNA chip in the aqueous solution. The formerspotting procedure is preferred The sample nucleic acid fragment ispreferably contained in the spotted aqueous solution in a molar amountof approximately 1 to 10⁴ per one mole of the DNA fragment fixed ontothe carrier.

[0087] In the DNA chip of the invention, the DNA fragments are fixedonto the solid carrier preferably in an amount of 10⁻²⁰ to 10 −12mol./mm² . In the preferred range, the spacer molecules are well fixedon the solid carrier at free spaces between the fixed DNA fragments,whereby the DNA fragments are well aligned vertically on the carrier andthe free spaces are covered with the spacer molecules having ahydrophilic group on the top. The hydrophilic groups on the free spacesare also effective to keep the sample nucleic acid fragment and thethread intercalator from their non-specific adsorption on the solidcarrier.

[0088] According to the experimental study made by the presentinventors, the masked DNA chip (namely, the DNA chip having the spacermolecules fixed between the DNA fragments fixed on a solid carrier) ofthe invention can be employed for extremely high sensitive quantitativedetermination of a sample nucleic acid fragment such as a molar amountof 10⁻²⁰ to 10⁻¹⁶ mol., preferably 10⁻²⁰ to 10⁻¹⁸ mole., per 1 mm² ofthe solid carrier. If a smaller solid carrier such as an extremely smallelectroconductive substrate is employed, increased high sensitivequantitative determination of a sample nucleic acid fragment such as amolar amount of up to 10⁻²¹ mole., per 1 mm² of the carrier may beaccomplished.

[0089] The DNA chip having on its surface the spotted aqueous solutionof a sample nucleic acid fragment is then allowed for stand for acertain period for performing incubation. In the incubation procedure,the thread intercalator is apt to be adsorbed non-specifically by freeDNA fragments of the DNA chip which have not participated in thehybridization with the sample nucleic acid fragment Such unfavorablenon-specific adsorption of the intercalator by the free DNA fragmentscan be obviated by adjusting a salt concentration of the aqueoussolution of the sample nucleic acid fragment 0.1 M or more, preferably0.1 to 1.0 M.

[0090] It is not advantageous that the DNA chip having been subjected tohybridization is washed to remove free thread intercalators This isbecause that, in the washing procedure, the thread intercalators fixedto the hybrid structure may be liberated.

[0091] The fixation of the electrochemically active thread intercalatorto the hybrid structure can be analyzed by measuring an electric currentflowing from or to the solid carrier (ie., electroconductive substrate)along the hybrid structure having the electrochemically active threadintercalator. The measurement of electric current can be performed byany of known methods such as cyclic voltamography (CV), differentialpulse voltamography (DPD), and potentiostat. The differential pulsevoltamography is most preferred

[0092] The present invention is further described by the followingexamples.

EXAMPLE 1

[0093] (1) Preparation of DNA Chip

[0094] A gold electrode (surface area: 1 mm²) was immersed in an aqueous2N sodium hydroxide solution for one hour, washed with water, andimmersed in conc. nitric acid. The nitric acid was stirred for 15 minThe gold electrode was then washed with super pure water and dried. Onthus treated gold electrode was spotted 1 μL of an aqueous solutioncontaining 20 mers of adenine having a mercaptohexyl group at its5′-terminal (HS-dA₂₀, in an amount of 10⁻¹⁴ mol/μL), and the electrodewas allowed to stand for 2 hours. The electrode was washed with superpure water to give a DNA chip. The preparation of HS-dA₂₀ was made inthe manner described in Japanese Patent Provisional Publication No.9-288080.

[0095] (2) Fixation of Spacer Molecules

[0096] Onto the DNA chip prepared in (1) above was spotted 1 μL of anaqueous 2-mercaptoethanol solution (1 mM). The spotted solution wascovered and allowed to stand for 2 hours. The DNA chip was then washedwith super pure water, to give the DNA chip of the invention havingspacer molecules on its electrode.

[0097] (3) Synthesis of Ferrocene-Containing Thread Intercalator

[0098] The below-illustrated ferrocene-containing naphthalene diimidewas synthesized in the manner described in Japanese Patent ProvisionalPublication No. 9-288080:

[0099] (4) Detection of Sample Nucleic Acid Fragment

[0100] A sample nucleic acid fragment, namely, 20 mers of thymine (dT₂₀,which was complementary to dA₂₀ fixed on the DNA chip) was prepared in amanner similar to that described in the above-mentioned publication.

[0101] Onto the DNA chip prepared in (2) above was spotted 1 μL of anaqueous solution of dT₂₀, and the DNA chip was allowed to stand at 25°C. for 30 min., for incubation.

[0102] An aqueous electrolytic solution [mixture of aqueous 0.1 M aceticacid/potassium acetate solution (pH 5.6) and aqueous 0.1 M potassiumchloride solution) containing 50 μm of the ferrocene-containingnaphthalene diimide (prepared in (3) above)) was placed in a thermostatcell maintained at 20° C. In the aqueous electrolytic solution wereplaced tri-electrodes composed of the DNA chip (working electrode), aplatinum electrode (opposite electrode), and a silver/silver chloridereferential electrode), and differential pulse voltamooraphy (DPV) wasperformed. Subsequently, a peak current at 460 mV was determined fromDPV data.

[0103] The above-mentioned detection procedures were repeated usingaqueous sample nucleic acid fragment solutions of 10⁻¹⁹ mol./μL and10⁻¹⁸ mol./μL, to determine peak currents at 460 mV from the obtainedDPV data.

[0104] The peak currents are graphically illustrated in FIG. 2 by theline connecting the circles.

[0105] The above-mentioned detection procedures were repeated forcomparison, using an aqueous sample nucleic acid fragment solutioncontaining non-complementary dA₂₀ fragment. The peak currents aregraphically illustrated in FIG. 2 by the line connecting the blanksquares.

[0106]FIG. 2 indicates that a linear relationships between the amount ofthe spotted sample nucleic acid fragment and the peak current isobserved in the 10⁻²⁰ to 10⁻¹⁸ mol. range in the use of the DNA chip ofthe invention. This means that an aqueous sample nucleic acid fragmentsolution is quantitatively analyzed at least in the range of 10⁻to 10⁻¹⁸mol per 1 mm² of the electrode surface.

EXAMPLE 2

[0107] (1) Preparation of PNA Chip

[0108] On a gold electrode (surface area: 2.25 mm²) having a mercaptogroup was spotted a phosphate buffer solution containing1,2-bis(vinylsulfonylacetamide)ethane, to form a free vinylsulfonylgroup on the electrode surface. On that surface was further spotted 1 nLof an aqueous solution of the below-illustrated PNA-thymine fragment(PNA-T₁₀, 50 nM solution). The spotted solution was allowed to stand forone hour at room temperature. The electrode was then washed withdistilled pure water to remove a free PNA-T₁₀, whereby give a PNA chip.The preparation of PNA-T₁₀ was made in the manner described in P. E.Nielsen et al., Journal of Americal Chemical Society, 114,1895-1897(1992), ibid., 114, 9677-9678(1992).

[0109] (2) Fixation of Spacer Molecules

[0110] Onto the PNA chip prepared in (1) above was spotted 1 μL of anaqueous 2-mercaptoethanol solution (1 mM). The spotted solution wascovered and allowed to stand for 2 hours. The PNA chip was then washedwith super pure water, to give the PNA chip of the invention havingspacer molecules on its electrode.

[0111] (3) Detection of Sample Nucleic Acid Fragment

[0112] The detection procedures of Example 1-(4) were repeated exceptfor replacing the DNA chip with the PNA chip produced in (2) above.

[0113] Almost the same detection results as those observed in Example1-(4) were obtained.

What is claimed is:
 1. A DNA chip comprising a solid carrier and aplurality of DNA fragments fixed onto the solid carrier at each one end,wherein a plurality of short chain spacer molecules having a hydrophilicmoiety at each one end are fixed at each another end onto a surface areaof the solid carrier having no DNA fragments thereon.
 2. The DNA chip ofclaim 1, wherein the solid carrier is an electro-conductive substrate.3. The DNA chip of claim 1, wherein the DNA fragments are fixed onto thesolid carrier in an amount of 10⁻²⁰ to 10⁻¹² mol./mm².
 4. The DNA chipof claim 1, wherein the hydrophilic moiety of the spacer molecule is ahydroxyl group.
 5. The DNA chip of claim 1, wherein the spacer moleculeis fixed onto the solid carrier through a mercapto moiety attached tothe end of the spacer molecule.
 6. The DNA chip of claim. 1, wherein thespacer molecule is derived from a compound selected from the groupconsisting of 2-mercaptoethanol, 3-mercaptoethanol; 6-mercaptoethanol,and N,N′-di(3-hydroxy-n-propyl)-imidazole-2-thione.
 7. A process forpreparing a DNA chip of claim 1 which comprises the steps of: applyingonto a solid carrier an aqueous solution of a plurality of DNA fragmentsdissolved or dispersed in an aqueous medium to fix the DNA fragmentsonto the solid carrier; and applying onto the solid carrier havingthereon the fixed DNA fragments an aqueous solution of short chainspacer molecules having at each one end a hydrophilic moiety and at eachanother end a moiety reactive to fix to the solid carrier.
 8. A methodof quantitative analysis of a nucleic acid fragment contained in asample liquid which is complementary to the DNA fragments of the DNAchip of claim 2, which comprises the steps of: adjusting theconcentration of the nucleic acid fragment in the sample liquid so thata droplet of the sample liquid applied to the DNA chip should contain10⁻²⁰ to 10⁻¹⁶ mol. of the nucleic acid fragment per 1 mm² of thesurface of the electro-conductive substrate of the DNA chip; bringingthe nucleic acid concentration-adjusted sample liquid into contact withthe DNA chip, whereby hybridizing the nucleic acid with the DNA fragmenton the DNA chip; bringing an electrochemically active molecule incontact with the hybridized nucleic acid and DNA fragment, wherebyattaching the electrochemically active molecule to the hybridizednucleic acid and DNA fragment; applying a potential to the DNA chip; andmeasuring an electric current flowing from or to the electro-conductivesubstrate through the attached electrochemically active molecule.
 9. Akit for conducting quantitative analysis of a nucleic acid fragmentcontained in a sample liquid which is complementary to the DNA fragmentsof the DNA chip of claim 2, which comprises the DNA chip of claim 2 andan electrochemically active molecule which is attachable to a hybridizednucleic acid and DNA fragment.
 10. A PNA chip comprising a solid carrierand a plurality of PNA fragments fixed onto the solid carrier at eachone end, wherein a plurality of short chain spacer molecules having ahydrophilic moiety at each one end are fixed at each another end onto asurface area of the solid carrier having no PNA fragments thereon. 11.The PNA chip of claim 10, wherein the solid carrier is anelectro-conductive substrate.
 12. The PNA chip of claim 10, wherein theDNA fragments are fixed onto the solid carrier in an amount of 10⁻²⁰ to10⁻¹² mol./mm².
 13. The PNA chip of claim 10, wherein the hydrophilicmoiety of the spacer molecule is a hydroxyl group.
 14. The PNA chip ofclaim 10, wherein the spacer molecule is fixed onto the solid carrierthrough a mercapto moiety attached to the end of the spacer molecule.15. The PNA chip of claim 10, wherein the spacer molecule is derivedfrom a compound selected from the group consisting of 2-mercaptoethanol,3-mercaptoethanol, 6-mercaptoethanol, andN,N′-di(3-hydroxy-n-propyl)-imidazole-2-thione.
 16. A process forpreparing a PNA chip of claim 10 which comprises the steps of: applyingonto a solid carrier an aqueous solution of a plurality of PNA fragmentsdissolved or dispersed in an aqueous medium to fix the PNA fragmentsonto the solid carrier; and applying onto the solid carrier havingthereon the fixed PNA fragments an aqueous solution of short chainspacer molecules having at each one end a hydrophilic moiety and at eachanother end a moiety reactive to fix to the solid carrier.
 17. A methodof quantitative analysis of a nucleic acid fragment contained in asample liquid which is complementary to the PNA fragments of the PNAchip of claim 11, which comprises the steps of: adjusting theconcentration of the nucleic acid fragment in the sample liquid so thata droplet of the sample liquid applied to the PNA chip should contain 10⁻²⁰ to 10⁻¹⁶ mol. of the nucleic acid fragment per 1 mm² of the surfaceof the electro-conductive substrate of the PNA chip; bringing thenucleic acid concentration-adjusted sample liquid into contact with thePNA chip, whereby hybridizing the nucleic acid with the PNA fragment onthe PAX chip; bringing an electrochemically active molecule in contactwith the hybridized nucleic acid and PNA fragment, whereby attaching theelectrochemically active molecule to the hybridized nucleic acid and PNAfragment; applying a potential to the PNA chip; and measuring anelectric current flowing from or to the electro-conductive substratethrough the attached electrochemically active molecule.
 18. A kit forconducting quantitative analysis of a nucleic acid fragment contained ina sample liquid which is complementary to the PNA fragments of the PNAchip of claim 11, which comprises the PNA chip of claim 11 and anelectrochemically active molecule which is attachable to a hybridizednucleic acid and PNA fragment.